Methodology is a major limiting factor in biochemical studies of carcinogenesis. No methods are available for establishing cause and effect between an initial, observed chemical lesion and development of a tumor. The goal of this research is to develop a direct method for introducing a potentially carcinogenic chemical change at a single, known preselected position in a DNA molecule. By studying the biological effects of this change, it should be possible to establish whether or not this particular change can lead to malignancy. Our approach is based on the total synthesis of DNA by combination of chemical and biochemical techniques, in such a way that a single, known chemical lesion can be introduced into a preselected position in the DNA sequence. By transfection experiments, one can then observe the phenotypic expression of this well-characterized lesion. In this way, specific chemical changes that are known to be produced in DNA by mutagenic and carcinogenic agents can be examined to see if a cause and effect relationship really exists. Although the method is general, it is testable only with bacteriophage phi X174 DNA at present. We will attempt to establish the feasibility of this approach by synthesizing a primer oligonucleotide containing a 5-bromouracil residue in the first glutamine codon of gene G. This will be used to prime the enzymatic synthesis of viral DNA. The phenotypic expression of this bromouracil residue, located at a known position in the viral DNA, will be tested by transfection experiments with suitable bacterial hosts. If successful, these experiments will allow us to observe the in vivo phenotypic expression of a single, known chemical lesion in the DNA. Once established, the methodology should be extendable to any covalent reaction product known to be produced by interaction of DNA with a mutagen or a carcinogen, providing the product can be introduced into the DNA primer by organic synthesis. This would enable us to test current theories of mutagenesis and carcinogenesis rigorously.